土壤宏基因组中抗草甘膦新基因的克隆与转化水稻的研究

EPSPS (5-烯醇丙酮莽草酸-3-磷酸合酶EC 2.5.1.19)是植物芳香族氨基酸和植物次生代谢产物生物合成中莽草酸途径的关键酶; 同时也是广谱性除草剂草甘膦的作用目标。本实验通过对草甘膦污染土壤宏基因组文库的建立及筛选, 成功克隆了一个新的草甘膦抗性的EPSPS基因(命名为soilEPSPS)。序列分析表明soilEPSPS基因全长1404 bp, 其编码的467个氨基酸中未涉及已公布专利中保护的氨基酸序列。原核功能验证表明该基因对草甘膦的耐受能力优于EPSPS CP4基因。将该基因与水稻Rubisco SSU引导肽相融合构建由actin启动子驱动的植物表达载体, 用农杆菌介导法实现了水稻的遗传转化。抗性再生植株的PCR和Southern杂交结果表明所获得的26株再生植株均为转基因阳性植株, 其中共有3个单拷贝转化事件。草甘膦抗性鉴定证明纯合体T₂代植株能够耐受高达500 mmol L^-1的草甘膦。本研究为转基因抗除草剂水稻新品种的培育奠定了基础。     

作物抗草甘膦转基因研究概况

草甘膦是一种非选择性除草剂,其作用机理主要是竞争性抑制莽草酸途径中5-烯醇丙酮莽草酸-3-磷酸合成酶(EPSPS)的活性。该合成酶是真菌、细菌、藻类、高等植物体内芳香族氨基酸生物合成过程中一个关键酶。自从1976年美国孟山都公司的草甘膦类除草剂-农达(Roundup)研制成功并得到广泛应用以来,作物抗草甘膦转基因研究成为抗除草剂基因工程研究的热点。随着抗草甘膦基因克隆的发展,抗草甘膦转基因作物也相继问世并大面积推广应用。对草甘膦作用机理、抗草甘膦基因(EPSPS编码基因)的研究、抗草甘膦作物遗传改良的策略及抗草甘膦转基因作物的推广应用情况进行综述,并简单分析了我国作物抗草甘膦转基因存在的问题及解决途径。     

转EPSPS基因抗草甘膦棉花的遗传分析

为分析转基因抗草甘膦棉花早代遗传情况,以花粉管通道法获得的26个转5-烯醇式丙酮酰莽草酸-3-磷酸合酶基因(EPSPS)抗草甘膦棉花转化事件为材料,以其背景亲本中棉所49为对照,喷施草甘膦后对转基因棉T1、T2分离比例进行考察。T1田间抗性鉴定结果表明,经卡方检测20个转化事件T1分离符合3∶1的分离规律,即外源基因插入1个位点;6个转化事件不符合1对基因的分离规律,出现了偏分离。T2田间抗性鉴定结果表明,通过花粉通管法共获得152个纯合株系,分别来源于25个转化事件;对T2不纯合株系继续进行分离比例的考察,发现来源于15个转化事件的57个株系符合3∶1的分离规律;此外卡方检测结果表明,每个转化事件都有不符合3∶1分离规律的株系,且其中10个转化事件没有符合3∶1分离规律的株系。表明通过花粉管通道法获得的转基因植株中外源基因的整合和遗传均较复杂。     

陆地棉EPSPS基因的克隆及其组织特异性表达分析

 5-烯醇式丙酮莽草酸-3-磷酸合成酶（EPSPS）是莽草酸途径中的一个重要酶,它是非选择性除草剂草甘膦的靶标酶,在高等植物中定位于叶绿体质膜上。根据EST拼接的序列设计引物,从陆地棉品种珂字棉312中获得了全长为1834 bp的cDNA序列,其最大可读框为1565 bp,编码521个氨基酸。陆地棉EPSPS基因与其它植物中同类酶在氨基酸水平上有广泛的同源性。通过与已知的其它高等植物叶绿体转运肽剪切位点比对,推断棉花EPSPS基因含有74个氨基酸叶绿体运输肽和447个氨基酸组成的熟蛋白。该酶具有保守的PEP结合位点及催化位点的特征序列。半定量分析表明,该基因产物广泛存在于棉花根、茎和叶等各组织中,在叶片中表达量较高。进一步扩增棉花核基因组获得了3344 bp的DNA序列,它包含8个内含子和7个外显子。棉花EPSPS基因的克隆为抗草甘膦棉花种质资源的创制提供了理论基础。     

肠杆菌20 aroA基因的克隆及功能分析

挖掘草甘膦抗性基因将有助于抗草甘膦遗传改良作物的培育。本研究分离了一株新型草甘膦抗性菌株肠杆菌20(Enterobacter.E20),该菌株在营养缺陷型培养基中能够耐受高达400 mmol/L草甘膦的胁迫,为充分发掘该菌株在草甘膦抗性方面的利用价值,对该菌株进行了全基因组测序(Gen Bank:CP012999.1)。分离克隆了肠杆菌20其EPSPS(5-烯醇式丙酮酰莽草酸-3-磷酸合酶)编码基因aroA20(WP_039261572.1),该基因序列长度为1 284 bp,编码427个氨基酸,序列比对分析显示AroA20具有Ⅰ型EPSPS典型的保守结构特征,系统进化分析显示AroA20与大肠杆菌K12的EPSPS具有最近的亲缘关系。利用原核表达系统鉴定了重组aroA20菌株在草甘膦胁迫下的耐受性,在转基因烟草中过表达了aroA20基因并鉴定了转基因植株的草甘膦抗性。研究肠杆菌20与其草甘膦的靶标EPSPS编码基因aroA20在草甘膦抗性上存在差异的分子机制将为深入理解生物体草甘膦抗性奠定研究基础。     

抗草甘膦棉花研究进展

抗草甘膦棉花最早于1997在美国释放,它是通过将编码抗草甘膦酶5-烯醇式丙酮酰莽草酸-3-磷酸合成酶基因导入珂字棉312而获得的抗性株.抗草甘膦棉花在美国的种植面积占棉花总种植面积从1997年的3.2%到增加到2005年的60%左右.在这近十年内,抗草甘膦棉花虽然得到了广泛的种植,但也存在着不少问题,其中最为突出的是草甘膦的施用方法和时期的不正确操作对抗草甘膦棉花的根系生长、育性和产量都产生了负面影响.这主要是因为:1、外界因素,包括草甘膦施用方法在内的其他栽培措施和不利的环境条件,如高温,对抗草甘膦棉花的产量及生长的影响;2、内部因素,抗草甘膦棉花不同器官对草甘膦的吸收量及抗草甘膦基因的表达量差异有关.因此,本文从抗草甘膦棉花对草甘膦的吸收分配、草甘膦的施用量和时期对抗草甘膦棉花根系生长、花药开裂及产量等方面综述了这些年来抗草甘膦棉花在应用过程中的问题,并且对今后抗草甘膦棉花育种方面作了展望.     

月季EPSPS基因的克隆及生物信息学分析

月季是园林绿化中常用的花卉,因此十分有必要对其抗性基因进行研究。5-烯醇丙酮酸莽草酸3-磷酸合酶(EPSP; 3-磷酸莽草酸1-羧基乙烯基转移酶; EC 2.5.1.19)为莽草酸途径中的关键酶,也是除草剂草甘膦的靶向酶。为了探究月季EPSPS基因的结构和功能,本研究利用月季基因组信息设计特异引物克隆得到月季的EPSPS基因,命名为RcEPSPS。经RT-PCR克隆后得到RcEPSPS基因的ORF全长为1 566 bp,共编码521个氨基酸,分子质量为55.19 kD,等电点为7.5,亲水系数为0.009。初步研究得知RcEPSPS属于EPSPS I型蛋白,与同属蔷薇科的野草莓的序列同源性极高。研究结果可为后续抗性基因的利用提供理论基础。     

黄瓜EPSPS基因的克隆及其组织特异性表达分析

黄瓜是世界上重要的蔬菜植物之一,对其抗性基因的研究十分必要。5-烯醇式丙酮酰莽草酸-3-磷酸合成酶(EPSPS)是莽草酸合成通路中关键的合成酶,也是除草剂草甘膦的靶向酶。为了研究黄瓜EPSPS基因的结构及功能,基于黄瓜基因组数据库信息,利用RT-PCR的方法克隆得到黄瓜的EPSPS基因,命名为CumEPSPS。克隆得到CumEPSPS基因的ORF全长1 581 bp,编码526个氨基酸,分子质量为56.28 kD,等电点为6.23。CumEPSPS蛋白属于Ⅰ型的EPSPS,与葫芦科的甜瓜和冬瓜的EPSPS蛋白序列高度同源。CumEPSPS基因在黄瓜的不同组织中均有表达,其中叶片中的表达量最高。黄瓜CumEPSPS基因的克隆及其生物信息学分析将为黄瓜抗草甘膦的研究提供理论依据。     

抗双链RNA依赖性蛋白激酶PKR基因表达载体构建及在玉米中的遗传转化

玉米病毒性病害和杂草严重影响其产量和品质。以pCAMBIA5300为基础载体,应用In-Fusion克隆技术构建了双价植物表达载体pCAMBIA5300-Ubi-PKR-CaMV35S-EPSPS,其中含有抗双链RNA依赖性蛋白激酶PKR基因和磷酸烯醇式丙酮酸莽草酸-3-磷酸合酶EPSPS基因,分别由玉米泛素Ubi启动子和花椰菜花叶病毒35 S启动子启动。以玉米种子黄化苗的茎尖分生组织为受体,用农杆菌介导法进行遗传转化,将抗双链RNA依赖性蛋白激酶基因PKR和抗除草剂草甘膦基因EPSPS导入玉米自交系掖478中,获得转基因植株及其子代。     

草甘膦生物抗性和生物降解及其转基因研究

草甘膦(N—phosphonomethyl—glycine，glyphosate)毒性作用机理是竞争性抑制莽草酸途径中的5-烯醇丙酮莽草酸-3-磷酸合成酶(5-enolpyruvyl—shikimate—3—phosphate synthase，简称EPSP合成酶)的活性。EPSP合成酶是植物和微生物体内芳香族氨基酸(包括色氨酸、酪氨酸、苯丙氨酸等)生物合成过程中的一个关键酶。该酶由aroA基因编码。抗草甘膦微生物或植物中EPSP合成酶基因的核苷酸序列在相同或相近位点发生了突变。将编码EPSP合成酶的突变基因导入大豆和烟草等作物中，均能获得转基因的抗草甘膦作物。草甘膦的生物降解途径主要有两条，C-N断裂生成氨甲基磷酸(AMPA)或C-P键断裂生成肌氨酸(sarcosine)，然后两种中间代谢物进一步代谢为磷酸、甘氨酸和二氧化碳等。     

1 株抗草甘膦棉花突变体草甘膦抗性的初步鉴 定简

 目的在于探明1株新的草甘膦抗性棉花突变体是否能用于发掘新的草甘膦抗性基因以及在农业生产中作为抗草甘膦种质的选育和利用的潜在价值。本文采用PCR的方法,在基因组和转录水平上排除了CP4-EPSPS基因对该突变棉株的污染;通过测定莽草酸含量,鉴定了该突变体草甘膦抗性的典型生理特征;通过盆栽试验研究了该突变体苗期对草甘膦抗性的表型。结果显示：不论草甘膦处理与否,突变棉株莽草酸的含量都没有显著的积累,在生理水平上显示出草甘膦抗性植株的显著特点;2叶期时草甘膦处理结果显示,突变棉株的草甘膦抗性表型同孟山都的品种对草甘膦的抗性表型基本一致。在基因组和转录水平的PCR检测结果都排除了突变棉株草甘膦抗性的获得是CP4-EPSPS基因污染造成的。结合对其他已报道的草甘膦抗性基因的类似排除,说明该抗性突变存在着自身特有的分子机理。     

Generation and Glyphosate Resistance Analysis of G10eve Transgenic Cotton

[Objective] Novel germplasm of cotton was generated for development of cultivars with high resistance to glyphosate. [Method] The glyphosate resistance gene G10eve was preliminarily analyzed using bioinformatics methods and introduced into 'Coker 312' via Agrobacterium-mediated transformation. The expression of G10eve was estimated by PCR (Polymerase chain reaction) and qRT-PCR (Quantitative real-time PCR). The content of shikimic acid and tolerance to glyphosate were determined in response to application of glyphosate to 'Coker 312' and transgenic plants. [Result] A total of 28 independent positive transgenic lines were obtained. The transformation efficiency and the rate of seedling differentiation were up to 49.3% and 40.6%, respectively. PCR and qRT-PCR analysis showed that G10eve was integrated into the cotton genome, and the G10eve expression level differed significantly among the transgenic lines. At the cotyledon stage, the transgenic cotton showed resistance to 8 mL·L^－1 glyphosate, whereas 'Coker 312' showed phytotoxicity to the 2 mL·L^－1 glyphosate treatment. No significant accumulation of shikimic acid was detected in transgenic plants before or after glyphosate treatment, which indicated the typical physical characteristic of glyphosate resistance. [Conclusion] Overexpression of G10eve in cotton improved resistance to glyphosate. Thus, novel germplasm of cotton resistant to glyphosate was generated.     

市售草甘膦除草剂对转基因抗草甘膦棉花幼苗生长的影响

 以转基因抗草甘膦棉花种质系G6-7和G6-8及其非转基因遗传背景亲本对照中棉所49为材料,研究了市售10%草甘膦水剂和95%的草甘膦粉剂对抗草甘膦棉花种子萌发和幼苗生长的影响。结果表明,中棉所49对于两种类型的草甘膦均很敏感,幼苗喷施0.2 mmol·L^-1 的10%草甘膦水剂和95%草甘膦粉剂均全部死亡。转EPSPS-G6基因抗草甘膦棉对95%草甘膦粉剂的抗性较好,但对10%草甘膦水剂的抗性较弱。就转基因棉花对两种剂型草甘膦的反应看,喷施2.4 mmol·L^-1的10%草甘膦水剂和95%草甘膦粉剂后,棉花幼苗全株鲜重、叶重、根重、下胚轴长、可溶性蛋白质增量和POD活性等各项指标在两种草甘膦制剂间的差异均达到显著水平。喷施12.0 mmol·L^-1 的10%草甘膦水剂,转基因抗草甘膦棉花的种子发芽率显著低于相同浓度的粉剂,两者之间的发芽率相差25%~75%。研究结果表明,10%的草甘膦水剂中可能含有对棉花幼苗生长发育有害的物质,影响抗草甘膦棉幼苗的生长,在转基因抗草甘膦棉花品种商业化应用时应谨慎使用。     

半野生棉与亚洲棉对草甘膦抗性评价及比较分析

采用叶片棉球定位鉴定法,初步鉴定并评价了半野生棉和亚洲棉对草甘膦的自然抗性,结果表明:参试材料抗性水平从高抗至高感分6级,极端抗性水平相差33.3倍(0.2%对0.006%)。其中,半野生棉对草甘膦的自然抗性分级丰富,抗性水平分布自高抗至高感;但大部分半野生棉不耐草甘膦,近86%(346份)的材料抗性水平处于低耐级别以下。而亚洲棉抗性水平都处于高抗至低耐的4个级别,抗性多样性较差,但整体抗性水平较高,70.3%(71份)表现为耐或以上级别。从半野生棉和亚洲棉中分别筛选到2份和5份高抗材料,半野生棉中有1份高感材料,亚洲棉中无对草甘膦敏感材料。本研究报道的极端材料为棉花草甘膦抗性遗传机制研究、新品种的培育、内源抗性基因的挖掘和感抗机理研究,奠定了良好的材料基础。     

Nitrogen Fertilizer and Its Residual Effect on Cotton Yield and Biomass Accumulation

[Objective] The optimum nitrogen rate of 300 kg·hm^－2 is well documented for cotton in the Yangtze River Valley, China. It can be reduced to 225 kg·hm^－2 without reducing yield in late sowing under high planting density. The aim of this study was to determine the possibility of further reducing the nitrogen application rate at the first flower stage, its residual effects and influence on cotton yield formation rule. [Method] A pot experiment was conducted with five nitrogen levels (120, 150, 180, 210 and 240 kg·hm^－2) in 2014, but only 180 kg·hm^－2(for studying nitrogen residual effect) in 2015, to study the cotton growth process, yield and its components and biomass accumulation. [Result] Nitrogen levels significantly affected the yield and biomass accumulation, but not the growth process and nitrogen residual effect. Maximum seed cotton yield (30.5 g·plant^－1), boll weight (3.8 g) and biomass accumulation, especially in the reproductive and vegetative organs, was recorded in the 180 kg·hm^－2 nitrogen treatment. In the rapid accumulation period, the proportion of biomass accumulation in the reproductive organs was the highest. [Conclusion] In a soil with medium fertility level, the application of 180 kg·hm^－2 nitrogen was optimal, because the strength of biomass accumulation in reproductive organs increased during the rapid accumulation period.     

转CP4-EPSPs基因大豆杂交后代对草甘膦的抗性水平与遗传背景的相关性

第一代抗草甘膦转CP4-EPSPs基因大豆GTS40-3-2是国际上应用时间最长、种植面积最大的转基因作物。本文以6份GTS40-3-2衍生的抗草甘膦转基因大豆新品系为亲本,配制4个杂交组合,利用抗性分级法和相对株高法鉴定杂交亲本及其F_(2:3)子代对草甘膦的耐受性差异,分析其抗性水平与遗传背景的相关性。结果表明,以1230 g a.i.hm~(–2)草甘膦喷施处理时,转基因亲本及其F_(2:3)子代的苗期生长受草甘膦抑制不显著,而当喷施浓度提高至3690 g a.i.hm~(–2)和4920 g a.i.hm~(–2)时则抑制作用显著。供试的6个杂交亲本中以ZLHJ06-1568、ZLHJ10-713和ZLHJ06-698对草甘膦的耐受性相对较强,而4个F_(2:3)组合中以ZLHJ10-713×ZLHJ06-698后代在草甘膦喷施后株高受抑制最小,对草甘膦耐受性最强。不同组合后代对草甘膦的耐受性普遍优于其双亲,呈现出杂种优势。各组合后代与亲本之间对草甘膦的耐受性均呈正相关,但由于亲本间互作效应的不同,导致后代抗性水平产生差异。本研究表明草甘膦抗性基因CP4-EPSPs在大豆中的表达水平与其遗传背景相关联,为利用转基因大豆新种质培育转基因大豆新品种过程中目标基因的定向选择提供了参考依据。     

Increased Night Temperatures During Cotton's Early Reproductive Stage Affect Leaf Physiology and Flower Bud Carbohydrate Content Decreasing Flower Bud Retention

Higher than optimum temperatures during cotton's (Gossypium hirsutum L.) growing season is a common occurrence in cotton‐growing areas around the world with negative consequences on productivity. According to climate projections, night temperatures are expected to increase more than day temperatures. The objective of this study was to monitor and record the effects of higher than optimum night temperatures during cotton's early reproductive stage on physiological (photosynthesis, respiration and membrane damage) and biochemical (leaf adenosine triphosphate (ATP) levels, as well as glutathione reductase (GR) content and soluble carbohydrate concentrations of the leaf subtending the flower buds. Number, dry weight, carbohydrate concentrations and GR levels of flower buds were also measured at the end of the experiment. Growth chamber experiments were conducted using cotton cultivar ST 5288 B2RF and treatments consisted of normal day/night temperatures (32/24 °C) and high night temperatures (32/30 °C) for 7 days at squaring (approximately 4 weeks after planting). The results indicated that high night temperatures had an immediate effect on leaf respiration rates and membrane damage by significantly increasing them compared to the control and a similar pattern was observed on leaf photosynthesis and ATP levels that were markedly decreased. Leaf GR levels were also substantially increased under conditions of high night temperatures, in contrast to flower bud GR content which remained unaffected. High night temperatures had a significant effect on leaf carbohydrate concentrations resulting in significant decreases in hexose, sucrose and starch levels. Nevertheless, flower bud sucrose content was substantially increased under conditions of high night temperatures, while hexose content was decreased and starch concentrations remained unaffected. A detrimental effect of elevated night temperatures on the number of flower buds per plant and on the dry weight of flower buds was observed, and it was concluded that high night temperatures had a negative effect on cotton flower bud production due to disruptions on flower bud carbohydrate metabolism as a result of the insufficient GR response.     

转Bt基因棉花各组织器官对棉铃虫抗性的研究

用４个国内转基因棉花品系（９５－１,２５２７,９５－６０,纯抗）和１个美国转基因棉花品种（ＤＨ３）进行抗虫性试验。结果表明,棉花不同组织的抗虫性不同,花瓣,苞叶和顶端叶抗虫性最强;国内转基因棉花的４个品系和美国ＤＨ３的抗虫性强弱没有显著差异;转基因棉花的蛋白毒性在棉铃虫体内具有一定的残效。     

TRV病毒介导的基因沉默体系在棉花中的建立及应用

以陆地棉CLA1基因为标记基因,利用烟草脆裂病毒(tobacco rattle virus,TRV)载体建立基于病毒介导的棉花基因沉默体系(virus-induced gene silencing, VIGS)。病毒RNA2的RT-PCR分析证明,棉花子叶接种TRV病毒后,该病毒可高效扩散到受体的根、茎、叶等器官。利用TRV-VIGS体系同时诱导34份不同来源棉花材料CLA1基因沉默,尽管不同材料间的抑制程度有差异,但均可有效抑制CLA1基因的表达,说明该体系在棉花研究中的广谱利用性。GhMAPKKK基因受黄萎病菌诱导表达, 接种后96 h表达量达到高峰。利用TRV-VIGS体系,成功抑制了棉花GhMAPKKK基因的表达,与对照株相比,抑制后的棉花植株接种黄萎病菌后更易感病,说明GhMAPKKK参与了棉花对黄萎病菌的抗性反应。具有广谱性、灵敏性和高通量等特点的棉花TRV-VIGS体系建立将加速棉花功能基因组研究进程。     

Differences of the Photosynthetic Properties and Nitrogen Accumulation between Island Cotton and Upland Cotton

[Objective] Under the same cultivation conditions, we explored the differences in photosynthetic characteristics between upland cotton and sea-island cotton, and their dry matter and nitrogen accumulation and distribution patterns during the blooming period, to provide a theoretical basis for irrigation and fertilization of island cotton. [Method] Using Xinluzhong 75, Xinluzhong 54, Xinhai 35 and Xinhai 48 as materials, we tested the net photosynthetic rate (P_n), leaf temperature, dry matter accumulation and nitrogen absorption and distribution characteristics under the same management conditions. [Result] After the flowering stage, when compared with island cotton, the P_n of upland cotton was higher, but the leaf temperature and light illumination were lower. However, the difference in effect of stomatal conductance (G_s) on P_n between island cotton and upland cotton was small. The dry matter accumulation of shoots and the daily accumulation of shoots and reproductive organs in the whole growth period were higher for upland than for island cotton, with a lower dry matter proportion of reproductive organs in the early growth period, and higher in the late period for upland than for island cotton. When compared with Xinhai 48, the initiation of rapid nitrogen accumulation of Xinluzhong 54 was later and its duration was shorter, but the maximum nitrogen accumulation rate was higher; however, the initiation of rapid accumulation time of reproductive organs was later, and its rapid accumulation duration was longer. [Conclusion] The dry matter output efficiency, accumulation in reproductive organs and nitrogen accumulation in the later growth period of island cotton were lower than those in upland cotton. Therefore, under production conditions, the proportions of water and fertilizer use should be increased for island cotton according to different regions.